Clinical and Molecular Effects of GnRH Agonist and Antagonist on The Cumulus Cells in The In Vitro Fertilization Cycle

Background: Gonadotropin-releasing hormone (GnRH) analogues have been extensively utilized in the ovarian stimulation cycle for suppression of endogenous rapid enhancement of luteinizing hormone (LH surge). Exclusive properties and functional mechanisms of GnRH analogues in in vitro fertilization (IVF) cycles are clearly described. This study was performed to evaluate clinical and molecular impacts of the GnRH agonist and antagonist protocols in IVF cycles. For This purpose, gene expression of cumulus cells (CCs) as well as clinical and embryological parameters were evaluated and compared between two groups (GnRH agonistand antagonist) during the IVF cycle. Materials and Methods: Twenty-one infertile individuals were enrolled in this study. Subjects were selected from two groups of GnRH agonist(n=10) treated patients and GnRH antagonist (n=11) treated individuals. The defined clinical embryological parameters were compared between the two groups. Expression of BAX, BCL-2, SURVIVIN, ALCAM, and VCAN genes were assessed in the CCs of the participants using the real-time polymerase chain reaction (PCR) technique. Results: The mean number of cumulus oocyte complex (COC), percentage of metaphase II (MII) oocytes, grade A embryo and clinical parameters did not show noticeable differences between the two groups. BAX gene expression in the CCs of the group treated with GnRH agonist was remarkably higher than those received GnRH antagonist treatment (P<0.001). The mRNA expression of BCL-2 and ALCM genes were considerably greater in the CCs of patients who underwent antagonist protocol in comparison to the group that received agonist protocol (P<0.001). Conclusion: Despite no considerable difference in the oocyte quality, embryo development, and clinical outcomes between the group treated with GnRH agonist and the one treated with antagonist protocol, the GnRH antagonist protocol was slightly more favorable. However, further clinical studies using molecular assessments are required to elucidate this controversial subject.


Introduction
The gonadotropin-releasing hormone (GnRH) agonis t and antagonis t protocols are extensively utilized in the ovarian s timulation cycle to inhibit the endogenous rapid increase in the luteinizing hormone (LH surge) levels. The unique properties and functional mechanisms of GnRH analogues in the in vitro fertilization (IVF) cycles are well defined (1).
GnRH agonis t have a longer half-life and higher potential than native GnRH. They initially s timulate pituitary gonadotrophs and production of follicle-s timulating hormone (FSH) and LH hormones, thereby cause an expected response of gonads (2). In contras t, GnRH antag-onis t immediately suppress pituitary gonadotropin in the competition with the GnRH receptor, thereby prevent early excitatory phase of agonis ts. Recently, there have been an increasing interes t in using GnRH antagonis t in control ovarian hypers timulation (COH). GnRH antagonis t have beneficial effects compared to the GnRH agonis t. Mos t notably they cause fewer follicles and lower daily usage of es tradiol, and thus lower incidence of ovarian hypers timulation syndrome (OHSS), a serious complication which eventually helps the reproductive treatment (3). However, it has been reported that GnRH antagonis t adminis tration is along with a reduced live birth rate and an increase in the risk of pregnancy loss, which might be the result of impaired implantation and lower es tradiol concentrations on the firs t day of COH (2). In addition to the pituitary, the role of GnRH in other tissues including ovary, uterus, and placenta have been demons trated in previous s tudies. Although the mode of action of GnRH and its analogues are well determined on the pituitary level, its role in the extra pituitary tissues is s till not fully unders tood (4).
GnRH receptors are present on the ovarian epithelial cells, granulosa cells (GCs), and cumulus cells (CCs). CCs are involved in the follicular development, maturity, and quality of the oocyte (5). There is a bidirectional paracrine communication between the CCs and oocytes during folliculogenesis (6). By secreting paracrine markers including growth differentiation factor 9 (GDF-9) and bone morphogenetic protein-15 (BMP-15), the oocyte induces CC gene expression to ensure its development and maturation (7). For this reason, optimal development and the quality of the oocyte can be evaluated by the CC gene expression as a non-invasive method (6). Versican (VCAN) and activated leukocyte cell adhesion molecule (ALCAM) are expressed in the CCs and contribute to the extracellular matrix (ECM) formation (8). VCAN, which is a proteoglycan, is expressed in the GCs after ovulation induction. VCAN is cleaved following LH surge by a precise molecular pathway and the cleaved VCAN, as the functional form, is observed in the COCs (9). Since important growth factor receptors are attached to this functional form, a change in the VCAN expression might also alter COC matrix properties during the oocyte maturation, ovulation, and fertilization (8). ALCAM is known as an ECM-related protein. Cell to cell and cell to matrix adhesion may be promoted by ALCAM in the reproductive tissues. ALCAM has been shown to be expressed in the epithelium and blas tocys ts and is involved in the implantation process (10). A significant association is reported between the expression of these genes and oocyte quality (11).
Moreover, it seems that apoptosis of CCs reduces the success rate in IVF (12) and the higher the incidence of apoptotic CCs, the lower the fertilization rate (13). The vital role of programmed cell death in different physiological events of reproduction is well es tablished. For ins tance, during folliculogenesis, the number of follicles in a follicular cohort primarily diminishes due to the apoptosis of GCs (14). SURVIVIN is a member of inhibitors of apoptosis proteins (IAPs) and has an important caspase inhibitory function (15). Critical functions of survivin in folliculogenesis and follicular development are not limited to apoptosis inhibition, but also this protein participates in the regulation of the mitotic spindle checkpoint (16). Follicular development or atresia are regulated by different hormonal and microenvironmental factors (17). AMH, GnRH, androgens, and apoptotic (BAX, P53, FOXO3) and anti-apoptotic (BCL-2, SURVIVIN) genes are identified as the follicular atretogenic factors (18). The anti-apoptotic role of BCL-2 agains t a variety of cell death-inducing factors has been proved in numerous s tudies. A correlation has been found between apoptosis ac-celeration and overexpression of BAX, as a pro-apoptotic agent (19). Therefore, the present s tudy was performed to examine the impact of GnRH agonis t and antagonis t on IVF cycles from clinical and molecular points of view. For this purpose, the oocyte quality, embryo development, CC gene expression, and pregnancy rate were compared between the two groups of patients who received GnRH agonis t or antagonis t throughout the IVF cycle.

Patients and s tudy design
In this s tudy, 21 eligible infertile women undergoing IVF cycle were chosen. This s tudy was conducted in Vali-e-Asr Reproductive Health Research Center, Tehran University of Medical Sciences (Tehran, Iran) from December 2014 to February 2016. S tudy was approved by Ethics Committee of Tehran University of Medical Sciences (IR.TUMS.VCR.REC.1396.2309). People who agreed to take part in this s tudy signed a consent form. Participants were divided into two groups of subjects, who received either GnRH agonis t or GnRH antagonis t. Subjects had an equal chance of being in both groups.
The inclusion criteria for the subjects were age<40 years and body mass index (BMI) <30 kg/m 2 . Furthermore, the cause of undergoing IVF was tubal factor infertility, and according to WHO criteria, partners had normal sperm parameters. The exclusion criteria were as follows: patients with ovarian dysfunction or other endocrinopathies, infertile couples with severe male factor infertility, poor responders, polycys tic ovarian syndrome (PCOS), and endometriosis.

S tudy size
The choices of sample size and s tudy duration were based on the primary outcome obtained from the s tudy of Danhua Pu 2011. A sample size of 11 with 80% power was achieved by a Two-Sided One-Sample t tes t, which can detect an effect size (i.e. mean difference) of 1.9 between the null hypothesis with no mean difference and the alternative hypothesis mean=-1.9 with an assessed s tandard deviation of 2.4 and alpha= 0.05. The sample size es timation was conducted using PASS 15 software.

S timulation protocols
Ten individuals were picked out from the GnRH agonis t treated patients that received triptorelin 0.1 mg/day subcutaneously (Decapeptyl, Ipsen, Italy) in the luteal phase of their preceding cycles based on a s tandardized long protocol. Following gonadotrophin inhibition, which was confirmed by transvaginal ultrasound, the patients received 150-225 IU recombinant FSH (rFSH) (Gonal-F, Merck Serono Laboratories, Switzerland) on the 3 rd day of their periods (20).
Eleven women in the GnRH antagonis t group were treat-ed with 150-225 IU/day rFSH subcutaneously beginning on the second day of their monthly periods followed by a single dose adjus tment from day 5 of the cycle. Each patient received 0.25 mg/day of cetrorelix (Cetrotide, Serono) on the sixth day of COH according to a fixed protocol (20).
To assess the ovarian response to the s timulation protocol, prior to the injection of the human chorionic gonadotropin (HCG) hormone, the follicle sizes were measured and clinical tes ts such as serum es tradiol and FSH concentration measurements and transvaginal ultrasounds were performed. Serum FSH and es tradiol concentrations were measured using immunoassay kits (CALBIOTECH, USA) with an automated multi-analysis sys tem.
A single dose of HCG (Gonasi HP 5000, AMSA, Italy) 10,000 IU was injected intramuscularly following the observation of at leas t three follicles with an optimal diameter of 18mm and serum es tradiol ≥0.40 nmol. Oocytes were picked up 34-36 hours after HCG adminis tration.

Evaluation of parameters
Embryological, clinical, and molecular variables were evaluated to compare the effects of GnRH antagonis t and agonis t. The pregnancy rate and the number of ovarian follicles were evaluated as clinical parameters.
The pregnancy rate was evaluated as the percentage of the subjects with positive βHCG tes t after receiving either the agonis t or the antagonis t protocol. The number of years that a woman was infertile was considered as the infertility duration. According to the cons tructor's ins tructions, serum prolactin concentrations were measured using an ELISA kit (Calbiotech, USA). The endometrial thickness and ovarian follicle count (number of follicles more than 18 mm) were measured using gynecological ultrasound.
The percentage of 7-cell embryos with less than 10% fragmentation (graded as A, Fig.1E) and the percentage of embryos with at leas t 7 cells having >10% fragmentation (graded as AB, Fig.1F) from the total number of fertilized oocytes on day 3 after insemination were assessed and compared between the two groups.
For the molecular inves tigation, expression of VCAN, ALCAM, SURVIVIN, BAX and BCL-2 genes were evaluated in the CCs.

Collection and isolation of cumulus cells
The retrieved COCs were washed several times in commercial human tubal fluid (HTF Lonza, Verviers, Belgium) in order to eliminate any blood cells, GCs, and debris contamination. Then, they were incubated in the fertilization medium for 5 minutes (Universal IVF medium, Medicul, Denmark). CCs samples were mechanically dissected less than 1 hour after OPU. Isolation of CCs was performed by washing these cells in the culture medium and centrifugation 10 minutes at 250 × g several times (21). Then, the cells were pooled and preserved by rapid freezing jus t after dissection and prior to the RNA extraction.

RNA extraction and real-time polymerase chain reaction
CC RNA extraction was performed using Arcturus Pico Pure RNA Isolation Kit (Applied Biosys tems, USA) based on the manufacturer's ins tructions (from ~ 4 ng pooled oocyte to 100 ng of CCs, and 3 repetitions for this experiment). DNase I (Fermentas, S t. Leon-Rot, Germany) was used three times to eliminate genomic DNA contaminations. The purified RNA was used for cDNA synthesis using oligo dT primers (Applied Biosys tems, Fos ter City, CA) prior to real-time polymerase chain reaction (PCR) (22).
The primers were designed to the human sequence of VCAN, ALCAM, SURVIVIN, BAX and BCL-2 genes using the Gene Runner (version 3) and Primer Express (version 3.05), and were blas ted in http://www.ncbi.nlm. nih.gov/BLAS t/. The primer characteris tics are presented in Table 1.
Real-time PCR was accomplished with the SYBR Green Reagent (Applied Biosys tems, USA) using ABI PRISM 7300 Analyzer (Applied Biosys tems, USA). The PCR cycle was repeated for 45-55 cycles. The Q-PCR reaction was carried out at leas t three times using specific primers. The quantification of 5 genes was evaluated using the comparison with the housekeeping gene, betaactin. Finally, 2 −∆∆CT technique was used for comparative quantification between the two groups.

S tatis tical analysis
Data were analysed using IBM SPSS S tatis tics software (version 25, IBM SPSS S tatis tics, Armonk, USA) and the graphs were drawn by GraphPad (Prism) (version 8, https://www.graphpad.com). Normality of the numeric variables was checked and confirmed by Kolmogorov-Smirnov tes t and measures of dis tribution including skewness and kurtosis were within ± 1.5 and ± 2, respectively. Data are presented as the mean (SD) and frequency (percent) for numeric normal and categorical variables, respectively. Comparisons of the variables between the groups were conducted by Independent Samples t tes t. The assumption of the homogeneity of the variances were assessed by Levene's tes t, and Welch correction was used when the assumption was not satisfied. For comparing the categorical variables between the two groups, Pearson Chi-square tes t with exact P value was utilized. In all analyses, a P<0.05 indicates s tatis tically significant.

Results
Clinical characteris tics of the woman in different groups are shown in Table 2. No significant difference was observed in the infertility duration, age, BMI and hormonal levels between the two groups. In addition, the number of the dominant ovarian follicles, endometrial thickness, and pregnancy rate were not different significantly between the groups.

Embryological assessments
In GnRH antagonis t group, the mean number of obtained COCs was higher than the GnRH agonis t group, which was not s tatis tically significant (P=0.14, Table 3). In order to compare the oocyte nuclear maturity, the percentages of MII, MI, and GV oocytes were calculated and compared between the groups. As shown in Table 3, MII percentage is clinically higher in the GnRH antagonis t group compared to the agonis t group (84.8 ± 20 vs. 78.6 ± 27.6, P=0.57). No considerable difference was found in the percentage of MI and GV oocytes between the two groups (12.6 ± 17.8 vs. 9.2 ± 16.2, and 5.6 ± 9.6 vs 5.5 ± 8.9, P=0.65 and P=0.99, respectively). Moreover, a s ta- .87) between the GnRH agonis t and GnRH antagonis t groups.

Discussion
This s tudy showed that BAX gene expression in the CCs of patients treated with GnRH agonis t was higher than those treated with GnRH antagonis t. Furthermore, mRNA expression of BCL-2 and ALCM genes were considerably greater in the CCs of the antagonis t group in comparison to the agonis t group. The gene expression of CCs in the individuals treated with assis ted reproductive technology (ART) have been evaluated in numerous previous s tudies (23). To bes t of our knowledge, this is the firs t s tudy to inves tigate the effect of GnRH analogues on CC gene expression.
The correlation between apoptosis of CCs and ART outcome has been demons trated in numerous s tudies (23). Clavero et al. (24) reported that the apoptosis rate of the GCs is not associated with the oocyte maturity, quality, and pregnancy outcomes. However, Lee et al. (25) found a s trong correlation between the apoptosis of CCs and poor oocyte quality. Moreover, up-regulation of pro-apoptotic genes and downregulation of antiapoptotic genes in the CCs of the non-early cleavage embryos have been previously described (26). It was shown that survivin plays an essential role in the function of GCs and the inhibition of apoptosis (15). In addition, a positive relationship has been observed between the SURVIVIN gene expression in the GCs and pregnancy rate (27). According to a s tudy by Assou et al. (28), the overexpression of BCL-2 is associated with pregnancy outcomes. The present s tudy indicated that the relative expression of BCL-2 is higher in the GnRH antagonis t group as compared to the agonis t group. Moreover, BAX was overexpressed in the GnRH agonis t group as compared to the antagonis t group. Furthermore, we found no positive correlation between the expression of apoptotic genes and oocyte quality, embryo development, and pregnancy outcome.
The effect of different protocols of GnRH analogues on the ART cycle is controversial (29). Similar results have been reported by Kara et al. (30) regarding the serum proges terone and es tradiol levels and the pregnancy rate. Prapas et al. (31) reported positive effects of GnRH antagonis t on the live birth rate as well as embryologic and clinical outcomes. Furthermore, higher quality of blas tocys ts have been noticed in the recurrent implantation failure (RIF) patients that received GnRH antagonis t compared to those receiving agonis t treatment (32). Contrary to the mentioned s tudy, de Souza Jordão et al. (33) revealed a higher total oocyte number and quality, more embryo development, higher implantation rate, and better pregnancy outcomes following GnRH agonis t adminis tration. A recent meta-analysis showed an equal pregnancy rate, endometrial thickness, live birth rate, and cancelation rate after the use of GnRH agonis t and antagonis t in normal-responder patients (34). Although the clinical and embryological results of our s tudy are not consis tent with the aforementioned articles, the molecular findings are compatible. This contradiction may be due to different and incomparable sample sizes.
Two of the five genes that were found to be expressed during oocyte maturation were analyzed in the present s tudy (ALCAM, VCAN) (8). A negative correlation was explained between VCAN expression level and the percentage of mature oocyte formation. Moreover, decreased VCAN expression was shown in the CCs of the subjects with mature oocytes (35). In our s tudy, a relatively lower expression of VCAN, which was not s tatis tically significant, was observed in the GnRH antagonis t group. ALCAM is known as an ECM-related protein. Cell to cell and cell to matrix adhesion may be promoted by ALCAM in the reproductive tissues. ALCAM has been shown to be expressed in the epithelium and blas tocys ts and it has an important role in the implantation process (36). A previous s tudy s tated expression of ALCAM in the CCs and GCs during the ovulatory response (37). Moreover, a positive correlation between the ALCAM expression and proper embryo cleavage has been indicated. It was also introduced as a promising new marker for non-invasive embryo selection (35). We also found a significantly higher ALCAM e xpression i n t he C Cs o f the GnRH antagonis t-treated group in comparison to the agonis t group.

Conclusion
Despite no considerable difference in the oocyte quality, embryo development, and clinical outcomes between GnRH agonis t and antagonis t, the GnRH antagonist protocol is more favorable considering our molecular findings. In fact, further molecular s tudies should be performed on this controversial subject to define the exact effect of GnRH analogues on the reproductive sys tem and to identify any advantage or superiority between the GnRH agonis t and antagonis t protocols.